Antibodies, or immunoglobulins, comprise two heavy chains linked together by disulphide bonds and two light chains, each light chain being linked to a respective heavy chain by disulphide bonds in a "Y" shaped configuration. The two "arms" of the antibody are responsible for antigen binding, and include regions where the polypeptide structure varies, these "arms" being termed Fab' fragments (fragment--antigen--binding) or F(ab').sub.2 which represents two Fab' arms linked together by disulphide bonds. The "tail" or central axis of the antibody contains a fixed or constant sequence of peptides and is termed the Fc fragment (fragment--crystalline). The production of monoclonal antibodies was first disclosed by Kohler and Milstein (Kohler & Milstein, Nature, 256, 495-497 (1975)). Such monoclonal antibodies have found widespread use as diagnostic agents and also in therapy.
Each heavy chain has at one end a variable domain followed by a number of constant domains. Each light chain has a variable domain at one end and a constant domain at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain (CH1). The constant domains in the light and heavy chains are not involved directly in binding the antibody to antigen. The light chain constant domain and the CH1 domain of the heavy chain account for 50% of each Fab' fragment.
The variable domains of each pair of light and heavy chains form the antigen binding site. The domains on the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, connected by three complementarity determining regions (CDRs) (Kabat et al, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1987)). The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs are held in close proximity by the framework regions and, with the CDRs from the other domain, contribute to the formation of the antigen binding site.
The human CD3 antigen consists of a minimum of four invariant polypeptide chains, which are non-covalently associated with the T-cell receptors on the surface of T-cells, and is generally now referred to as the CD3 antigen complex. It is intimately involved in the process of T-cell activation in response to antigen recognition by the T-cell receptors.
All CD3 monoclonal antibodies can be used to sensitise T-cells to secondary proliferative stimuli such as IL1 (interleukin 1) and IL2 (interleukin 2). In addition, certain CD3 monoclonal antibodies are themselves mitogenic for T-cells. This property is isotype dependent and results from the interaction of the CD3 antibody Fc domain with Fc receptors on the surface of accessory cells.
Rodent CD3 antibodies have been used to influence immunological status by suppressing, enhancing or re-directing T-cell responses to antigens. They therefore have considerable therapeutic potential in the human for use as an immunosuppressive agent, for example for the treatment of rejection episodes following the transplantation of renal, hepatic and cardiac allografts. However their value is compromised by two main factors. The first is the antiglobulin response evoked due to the xenogeneic nature of the antibody. The second is the "first dose" syndrome experienced by patients following the initial administration of the antibody. The symptoms, which range in severity from fever and chills to pulmonary edema, and which in rare cases can cause death, are caused by the elevated levels of circulating cytokines associated with CD3-antibody induced T-cell activation. This phenomenon requires the cross-linking of the CD3 antigen on the surface of T-cells to accessory cells through Fc receptors; such proliferation does not occur with F(ab').sub.2 fragments of CD3 antibodies.
The first problem can be addressed by re-shaping or "humanising" the variable region genes of antibodies and expressing them in association with relevant human constant domain genes. This reduces the non-human content of the monoclonal antibody to such a low level that an antiglobulin response is unlikely. Such a reshaped antibody with a binding affinity for the CD3 antigen complex is described in UK Patent Application No. 9121126.8 (published as GB 2249310A) and its equivalents (European Patent Application No. 91917169.4, Japanese Patent Application No. 516117/91 and U.S. patent application Ser. No. 07/862,543).
There remains however the problem of the first dose response when these antibodies are used in therapy. Aglycosylation of antibodies has been described to reduce their ability to bind to Fc receptors in vitro in some cases. However, it is not predictable that this will be true of all antibodies, particularly in vivo, and aglycosylation may result in the introduction into the antibody of novel and unpredictable properties including novel Fc binding characteristics causing other undesirable effects. It is also possible that other undesirable properties not associated with Fc binding may be introduced to the antibody.
Moreover, it is of course of vital importance that aglycosylation is not accompanied by the loss of certain desirable features of Fc binding in addition to the loss of the undesirable features such as those attributable to the first dose response.